Yield optimization, microbial load analysis, and sensory evaluation of mungbean (Vigna radiata L.), lentil (Lens culinaris subsp. culinaris), and Indian mustard (Brassica juncea L.) microgreens grown under greenhouse conditions

Microgreens have been used for raw consumption and are generally viewed as healthy food. This study aimed to optimize the yield parameters, shelf life, sensory evaluation and characterization of total aerobic bacteria (TAB), yeast and mold (Y&M), Escherichia coli, Salmonella spp., and Listeria spp. incidence in mungbean (Vigna radiata (L.) Wilczek), lentil (Lens culinaris Medikus subsp. culinaris), and Indian mustard (Brassica juncea (L.) Czern & Coss.) microgreens. In mungbean and lentil, seeding-density of three seed/cm2, while in Indian mustard, eight seed/cm2 were recorded as optimum. The optimal time to harvest mungbean, Indian mustard, and lentil microgreens were found as 7th, 8th, and 9th day after sowing, respectively. Interestingly, seed size was found highly correlated with the overall yield in both mungbeans (r2 = .73) and lentils (r2 = .78), whereas no such relationship has been recorded for Indian mustard microgreens. The target pathogenic bacteria such as Salmonella spp. and Listeria spp. were not detected; while TAB, Y&M, Shigella spp., and E. coli were recorded well within the limit to cause any human illness in the studied microgreens. Washing with double distilled water for two minutes has shown some reduction in the overall microbial load of these microgreens. The results provided evidence that microgreens if grown and stored properly, are generally safe for human consumption. This is the first study from India on the safety of mungbean, lentils, and Indian mustard microgreens.


Genotypes used and growing conditions
To optimize the seeding density, harvesting stage, and marketable yield, seeds of 20 mungbean and lentil genotypes each and two Indian mustard genotypes (Table 1; Fig 1) were sown in three replications with different seeding-density: two-seed/cm 2 , three-seeds/cm 2 , and fourseeds/cm 2 for mungbean and lentil, while six, eight, and ten seeds/cm 2 for Indian mustard. The selected mungbean and lentil genotypes were very diverse for several parameters including antioxidant activities and the mineral profiles [3]. In addition, PDZM31 (Pusa Double Zero Mustard-31) is the first double zero (erucic acid <2% and glucosinolates <30ppm) mustard variety of India [32], while PM28 is a short duration variety. Microgreens are commercially produced under partially controlled conditions on different growing-medium like cocopeat or a combination of cocopeat, vermiculite, and sand. In addition, to avoid any variations (in quality of the produce) due to the growing conditions (temperature, photoperiod, etc.), the genotypes were grown under partially controlled conditions in the National Phytotron Facility, IARI, New Delhi which is located at the latitude, longitude, and altitude of 28.6412˚N, 77.1627˚E, and 228.61 m AMSL, respectively. The desired temperature was maintained for mungbean (28/26˚C), lentils (21/18˚C), and Indian mustard (21/18˚C) along with a 10:14 h of day and night cycles.
Freshly harvested seeds (Table 1) were obtained from the Division of Genetics, IARI, New Delhi having more than 90% germination. The seeds were surface-sterilized at room Table 1. Genotypes, seed-weight, and moisture content in the studied lentil, mungbean, and Indian mustard microgreens. temperature (24˚C) for one minute in 1% sodium hypochlorite (NaOCI) solution and rinsed twice with sterile water [33] and then sown in plastic trays (38×28×6 cm) in three replicates. The autoclaved (120˚C, 120 Pa, 90 min) growing-medium consisted of coco peat: vermiculite: sand (2:1:1) was used for growing these microgreens. Based on the growth rate, harvesting was performed at different durations for mungbean (after 5 th , 7 th , and 9 th day), lentil (after 7 th , 9 th , and 11 th day), and Indian mustard (after 6 th , 8 th and 10 th day) microgreens. Microgreens were harvested using ethanol-cleaned scissors by cutting the stem approximately 1.0 cm above the growing medium and were immediately weighed using analytical balance to determine the total fresh weight (FW). Afterward, these were dried in hot air GenLab vertical oven (40˚C for 72h), then weighted and kept in an airtight container for further biochemical analysis. The moisture content was calculated as per the equation:

S. No. (a) Mungbean microgreens (b) Lentil microgreens
Where, Initial weight (g) = Weight of fresh microgreens after harvesting; Final weight (g) = Weight of microgreens after 72h of drying at 40˚C

Microgreens microbial load analysis
The incidence of total aerobic bacteria (TAB), yeast & mold (Y&M), Salmonella spp., Shigella spp., Listeria spp., and E. coli were assessed for both unwashed (freshly harvested) and washed samples of mungbean (genotypes MH-810, MH-318, PS-16), lentil (genotypes K75, L4594, L830), and Indian mustard (genotypes PM28, PDZM31) microgreens as obtained from the partially controlled conditions. The washing was performed using double distilled water for two minutes and then samples were air-dried in the laminar airflow (Svision, India). The mungbean, Indian mustard, and lentil microgreens were harvested on 7 th , 8 th , and 9 th day of sowing, respectively, and were stored in zip lock bags at 4˚C and 1.0 g tissue was used to study the microbial load at 1 st , 2 nd , 4 th , 8 th , and 12 th day (day of harvest was considered 1 st day). Microbial growth was assayed following the standard protocols [34,35]. A 1.0 g sample was incubated in 10.0 mL sterile phosphate-buffered saline (PBS, 10x solution from Sigma Aldrich, USA) and vortexed (15 min). Plating of serially diluted samples (1.0 mL) was done on different agar plates. TAB population was identified by plating samples on nutrient agar (NA) supplemented with Amphotericin b (5.0mg/mL; an anti-fungal agent) and incubated at 37˚C for 24-48h. Y&M enumeration was performed by plating samples on potato dextrose agar (PDA, Merck, Germany) supplemented with 50.0 mg/mL Chloramphenicol and incubated at 25˚C for 48 to 72h.
Salmonella and Shigella were recorded (based on their colony morphology) by plating the samples on Xylose Lysine Deoxycholate agar (XLD, Merck, Germany) supplemented with Amphotericin b (5.0mg/mL) and incubated in dark at 35±2˚C for 24 h. Listeria spp. was identified by plating the samples on Chromed Listeria Agar (Merck, Germany) supplemented with Nalidixic acid (13.0mg/mL), Ceftazidime (10.0mg/mL), and Amphotericin b (5.0mg/mL) and incubated at 37˚C for 24 h. E. coli O157:H7 population was identified by plating the samples on Sorbitol MacConkey agar (Merck, Germany) and incubated at 37˚C for 24 h. Each microbial count was determined as the mean of three measurements and the result was expressed as log CFU per g of tissue.

Microgreens shelf life and sensory evaluation
For shelf life and sensory evaluation, two genotypes each of mungbean (MH810 and MH318), lentil (L830 and K75), and Indian mustard (PM28 and PDZM31) microgreens were harvested at the optimum stage and stored in food-grade linear low-density polyethylene (LLDPE) bags to avoid cross-contamination [30]. For mungbean and lentils, the genotypes having largest and smallest seed sizes were selected. The LLDPE bags are of 16×12 cm size (8.0 g per bag) and 51μ thickness. The samples were then stored in three replications at 4˚C (in dark) for different durations. A panel of seven semi-trained judges (aged 24-45 years) from the IARI, New Delhi (India) performed the sensory evaluation [36]. Sensory evaluation (color & appearance, aroma, taste, and overall acceptability) was performed after 2 nd , 4 th , and 6 th day of storage using a 10-point hedonic scale (10 = like ultimate, 9 = like extremely, 8 = like strongly, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly; 3 = dislike moderately, 2 = dislike strongly, and 1 = dislike extremely [34]. A score of 6 was considered as the limit of salability [37]. A sample size of 2.0 g of each microgreen was used for evaluation.

Electrolyte leakage analysis
The electrolyte leakage of freshly harvested and stored microgreens was measured to find the possible tissue deterioration during storage. For this, 20.0g microgreens sample (from each replicate) was dipped in 400 mL deionized double distilled water (at 20˚C) and gently shaken for 30.0 min. The solution conductivity (μs/cm) was then measured using a conductivity meter (Orion 4-star portable pH/conductivity meter; Thermo Electron Corporation, U.S.A.) by dipping the probe in the sample solution [29,38].

Statistical analysis
The experiments were conducted thrice and the results were presented as mean±SD. One-way analysis of variance (ANOVA) was performed using SPSS11.5 to compare the groups, and Pearson's correlation test was used to assess the correlation between means. The mean comparison was performed using Tukey's test and a P�0.05 was regarded as significant.

Results and discussion
There has been a growing interest in promoting a healthy lifestyle including consumption of nutritious and quality foods, and microgreens offer a very good option [27,[38][39][40]. However, growing conditions of microgreens affect their overall yield, while their storage for some time may facilitate the growth of certain harmful microorganisms having food safety risks. Thus, this study was aimed to find the optimum growth parameters, sensory details, and existing microbial load during storage of mungbean, lentil, and Indian mustard microgreens.

Optimization of microgreens yield
The growing medium is very crucial for proper germination, growth of microgreens and its physical properties including porosity and water holding capacity [41]. Three combinations of growing medium consisting of cocopeat: vermiculite: sand in the ratio of 1:1:1, 2:1:1, and 2:2:1 was studied and 2:1:1 medium was found best in terms of water holding capacity. For this growing-medium combination, we generally need only 1-2 light irrigation when microgreens were grown in plastic trays (without holes) under greenhouse conditions [42]. The trays were placed on a leveled surface on benches [24] and two most common factors affecting the total yield include seeding density and plant growth. Also, harvesting microgreens at the right stage is the key production strategy, since the time from sowing to harvesting varies greatly from crop to crop [3,42].
Optimum seeding density is very specific to the crop species and is generally based on mean seed weight and germination (%) [42]. The yield of microgreens in this study showed an increasing trend with increasing seeding-density (Table 2a and 2b, Fig 2a). But, once it crossed the optimum seeding density, the marketable quality of microgreens got deteriorated. In Table 2. Lentil and mungbean microgreens yield of twenty genotypes each at different seeding densities.

S. No. Genotype (a) Mungbean
Yield (g/m 2 ) Genotype (b) Lentil Yield (g/m 2 )  Table 2a and 2b and Fig 2a. For mungbean, and lentils 3-seed/cm 2 was found optimum, while for Indian mustard it was 8-seed/cm 2 . Any increase in the seeding density beyond

PLOS ONE
Yield optimization, microbial load and sensory evaluation of mungbean, lentil and brassica microgreens optimum resulted in poor marketable-quality produce. Excessive plant stand resulted in undesirably elongated shoots (due to more congestion and competition). Higher seeding density also hampered air circulation, favorable for fungal growth [43]. In addition, increase in seeding density result in higher seed cost. Day of harvesting is also equally important for reaping the best marketable-quality yield. On 5 th to 9 th day, the mungbean microgreens yield ranged from 1773.87±30.32 to 2645.06 ±52.60 g/m 2 ; while on 7 th to 11 th day, the lentil microgreens yield ranged from 840.14±45.87 to 1411.29±77.66 g/m 2 ( Table 2) and the Indian mustard microgreens yield on 6 th to 10 th day ranged from 1070.66±35.13 to 1346.87±27.27 g/m 2 (Fig 2b).
Different microgreens species have different harvesting stages to achieve their marketable hypocotyl length and leaf area to reap maximum economic benefit. The mungbean, Indian mustard, and lentil microgreens have different growth rates and under studied conditions; 7 th , 8 th , and 9 th day, respectively were found optimum for harvesting (Table 3; Fig 2b). Even though the overall yield recorded was higher during the later stages of harvesting, the quality of microgreens got deteriorated (S1 Fig). Additionally, significant genotypic differences for yield were observed in the studied microgreens. Similarly, the microgreens yield was recorded as 659 g/m 2 in Brassica oleracea L. and 1548 g/m 2 in Cichorium intybus L. [27]. Also, the optimum days to harvest for radish microgreens were 7 th day, arugula-9 th day, and red cabbage-11 th day under specific growing conditions [28]. A very high correlation was recorded between mean seed weight and yield in both mungbean (r 2 = .73) and lentil (r 2 = .78) genotypes. As we used only two Indian mustard samples with nearly the same seed weight, the correlation analysis could not be performed.

Microbial counts
In many countries, various microbial outbreaks have been reported mainly due to the consumption of contaminated sprouts [19][20][21]. Thus, it becomes imperative to monitor and evaluate the microbial load in the microgreens too. Among different studied microbes, we have recorded the growth of Y&M, TAB, Shigella, and E. coli (O157:H7) in the studied microgreens. The target pathogenic bacteria Salmonella spp. and Listeria spp. were not detected in any of the tested samples. On contrary, Bergšpica et al. [11] have detected the presence of Listeria innocua in the radish and sunflower microgreens, and Salmonella spp. in sunflower microgreens. In general, Listeria innocua is considered to be a non-pathogenic Listeria species [44].
Washing of microgreens was done using double distilled water for 2-minutes and results of both washed and unwashed samples for the growth of various microbes in mungbean was found comparable to lentil and mustard microgreens (Table 4). On contrary, Chandra et al. [29] reported a very high value for the TAB count (7.8 logCFU/g) of unwashed cabbage microgreens; which after washing get reduced to 7.2 logCFU/g. Washing has shown a significant (P�0.05) reduction in the TAB (2.6 to 3.4 logCFU/g) and Y&M (1.1 to 2.2 logCFU/g) over fresh-cut beetroot samples [45]. Survival of E. coli O157:H7 was reported on radish [46], arugula, kale, lettuce, and mizuna microgreens [47]; while Di Gioia et al. [48] reported microbial growth on brassica microgreens. Inoculation of seed and irrigation water with Shiga toxinproducing E. coli (STEC) has resulted in the growth of bacteria on eight microgreens species [49].
No significant difference was recorded in the overall microbial load between the microgreens of different studied genotypes (Table 4a-4c). In general, an increasing trend was recorded for various microbial counts when samples were stored at 4˚C from 1 st day to 12 th day (S2 Fig). Similarly, Chandra et al. [29] also recorded an increasing trend in the microbial population during storage of Chinese cabbage and beetroot samples [45].
In mungbean microgreens, washing significantly decreased the load of Shigella spp., whereas, amicrobial count (TAB, Y&M, and E. coli) did not show any significant decreasing effect (Table 4a). However, in lentil microgreens, washing significantly reduced the overall load of Shigella spp. and E. coli; but TAB and Y&M count did not show much reduction (Table 4b). This means that the survival and growth need of Shigella spp. and E. coli are different from that of aerobic bacteria and Y&M in the microgreens, as also recorded by Chandra et al. [45]. In mustard microgreens, although washing reduced overall microbial load, it was not very significant (Table 4c).
Washing of harvested microgreens has been practiced to remove the attached soil particles, to reduce the initial microbial load, and also for clean packaging. However, washing reportedly creates humid environmental conditions suitable for microbial growth, thus necessitating careful removal of excess moisture without causing any damage to the greens [18]. Relatively faster loss of shelf life was reported for the washed radish [31] and buckwheat microgreens over unwashed microgreens; which could be due to the damage caused during washing and dewatering, and also the presence of excess moisture in washed microgreens packages [30].
Microgreens are prone to bacterial internalization as the bacteria present in the seeds can become part of endophytic microflora [50]. Also, during germination, the bacteria present in the rhizosphere are attracted by the seed exudates and may enter through the germinating radicals or secondary roots [50]. Therefore, once contaminated, it is nearly impossible to eliminate the microbes from the living plant system. Thus, sanitization or washing of harvested microgreens may not be a very effective control strategy. In addition, microgreens being very delicate are quite prone to the damage caused by any such treatments [18]. A much lower value of microbial load in the present experiments could be due to the good agronomic practices used during the growth of the microgreens including the use of freshly harvested seeds, seed treatment, autoclaving of growing media, and use of alcohol cleaned scissors while harvesting the microgreens.

Microgreens shelf life and sensory evaluation
Since microgreens are very tender, thus are extremely vulnerable to dehydration and quality deterioration. Therefore, to maintain the quality and shelf life of microgreens, proper refrigeration and packaging become extremely crucial [18]. At the time of harvesting, microgreens have a very high respiration rate [29] and can be stored comfortably for nearly a week time at <5˚C [30,31]. Immediately after the harvest, microgreens should be washed and cooled (1-5˚C) [26] or this can be marketed in trays with growing-medium [24]. Thus, two genotypes each of mungbean (MH810 & MH318), lentil (L830 and K75), and Indian mustard (PM28 & PDZM31) were used for the shelf life and sensory evaluation. These were stored at 4˚C for 6 days and analyzed at 1 st , 2 nd , 4 th and 6 th day of storage. The visual appearance of microgreens declined gradually as the storage time increased under cool (4˚C) conditions (S1 Fig). Mungbean and Indian mustard microgreens showed nearly 4-day shelf life, while lentil microgreens could be used till 6 th day of their storage in 51μ thick LLDPE zip-lock bags (16×12 cm) at 4˚C conditions. On contrary, based on visual parameters, the shelf life of arugula, radish, and red cabbage was recorded as 14, 21, and 14 days, respectively at 4˚C; whereas, at 10˚C this was 7, 14, and 7 days, respectively [28]. Till 4 th day of the storage at 4˚C, all the studied sensory parameters such as color and appearance, aroma, taste, and overall acceptability of the studied microgreens showed the hedonic score of >6, which was considered as the limit of salability [37]. However, on the 6 th day of storage, a drastic reduction in all the sensory parameters of mungbean and mustard microgreens was recorded. Interestingly, lentil microgreens showed >6 hedonic scores for all the studied sensory parameters, even on 6 th day of its storage (Table 5a-5c). This could be due to relatively less moisture content in the lentil microgreens over mungbean or Indian mustard microgreens. In general, the moisture content in mungbean, lentil, and Indian mustard microgreens ranged from 90.01±1.44 to 93.06±1.07, 82.69±2.48 to 85.95±1.01, and 89.83% to 90.5%, respectively. An inverse relationship was found between the moisture content and the shelflife (and sensory qualities) in the studied microgreens. Many reports underlined the importance of temperature in prolonging the overall post-harvest shelf life of various fresh-cut products including microgreens [51][52][53]. A slower respiration rate at low temperature can be directly correlated with the lower rate of cellular metabolism and cause a direct effect on visual microgreens quality and hence increased self-life [28].

Electrical conductivity (EC) of washed and unwashed microgreens
EC can be associated with the overall quality and shelf life of microgreens and is used as an indirect measure of the same [37]. With increasing storage duration, EC showed an increasing trend in both washed (with sterile double distilled water for 2.0 min) and unwashed microgreens over fresh samples (Fig 3). EC was recorded more for the mungbean microgreens, especially at 4 th day (6.97 μs/cm) and 6 th day (15.63 μs/cm) of storage over lentil (3.37 & 6.57 μs/ cm) or Indian mustard (6.4 & 14.0 μs/cm) microgreens, respectively. Relatively less EC was recorded for 4 th and 6 th -day samples under 4˚C storage; while it was a bit more for washed samples for 2 nd day. Cell surface damage caused during washing treatment might have got repaired by 4 th day of storage. Interestingly, 6 th day of storage showed a sudden rise in EC for all the microgreens (Fig 3). Conductivity values showed a positive association with the storage duration of the studied microgreens.
On a similar note, the EC values showed a 7-fold increase (over initial value) for the tap water-washed Chinese cabbage microgreens, until the end of storage (9 th day), when packed in polypropylene (PP) film [29]. Similar observations were also recorded for fresh-cut cilantro [54]. An increase in EC values under storage may be due to the irreversible membrane damage and accumulation of CO 2 from respiration [29]. On contrary, a decreasing trend in the electrolyte leakage was recorded for broccoli microgreens for various washing treatments and O3 washing (180s) under 09-day storage conditions [38].

Conclusions
Rapid growth cycle, limited space requirement, rich flavor, diverse color, and highly economic produce makes microgreens a nutrient alternative that may contribute to the nutritional security of a large population. To the best of our knowledge, no study about the yield optimization and microbial aspects of mungbean, lentil, and Indian mustard microgreens has been reported so far from India. The use of good agricultural practice is the key to manage the microbial contamination of the growing microgreens [55]. More scientific information should be generated for various microgreens to eliminate the possibility of microbial contamination through seed, grow-media, grow-trays, and harvesting implements. In addition, post-harvest care such as harvesting at an optimum stage, proper sanitation, and maintenance of optimum temperature and humidity will help in longer storage and reduced risk of human pathogen contamination [12,18,56]. Thus, if grown and stored properly, there is no major risk of microbial illness from any kind of microgreens consumption. The success of microgreens technology will largely depend on the collective and collaborative efforts from the industry and researchers in the food-chemistry, biochemistry, genetics, and human nutrition working to enhance the yield and quality. This is the first such study from India, which included the microgreens of mungbean, lentil, and Indian mustard. Interestingly seeds of studied crops are readily available in any Indian kitchen, and we hope that the results will help in the popularization of these microgreens even at household levels.